Multiple mechanisms of neutralization of animal viruses

70

T I B S 1 2 - February 1987

m o u r laboratory was supported by N I H grant GM30726 References I Rushlow K E Orozco E M Jr Lnpper C and Halhck R B [1980)J Bml Chem 2~S, 3786-~792 2 Bnal J-F Laulhere J-P and Mache. R (1979) Fur J Bmchem 98 285-292 3 Sun E Shapiro. D R Wu B W and Texan K K (1986)Plant Mol B:ol 6 429439 4 Grutssem W Greenberg B M Zurawskt G Prescott D M and Hallnck R B 0983) Cell35 815-828 Gruissem W Elsner-Menzel C Ldtshaw 8. Ndrlla J O,Shdffer M A and Zurawskn G (1986) Nuclei{ AeMs Res [4 7541-7.~56 6 Link G (1984)EMBOJ ~ [697-1704 70rozco E M Jr Mullet J E and Chua N- H ( 1985) Nu dew Acrds Res 13 128~- I ~02 8 Crousc E J Schmnll J M andBohnert H-J 0985) Plant ~1o1 Blol Rep ~ &'~--89 9 Shmozak~ K el al 0986) EMBO J 5 2(t4~2049 10 Wh~tfeld P R and Bonomlev W (1983)

Annu Rev Plant Phvswl 34 279-310 I I Hanley-Bowdom L. Orozco E M, Jr and Chua N-H (1985) in Molecular Biology o] the Pholos)r"wuc Apparatrts (Arntzen C . ~]ogorad. L Bomtz, S and Stembaek K. ed ),pp 311-318 Cold Spnng Harbor Press 12 Buyer S K and Mullet J E (1986)Plant Mol B~ol 6 229-243 I3 Bradley, D and Gatenby, A (1985) EMBOJ 4, 3641-3648 14 Hanley-Bowdom. L Orozco. E M Jr and Chua N-H 0985)Mol Cell Btol 5. 27332745 15 Grmssem. W and Zurawskt G 0985) EMBOJ 4 3375-3383 16 Grmsscm. W and Zurawskl O (1985) EMBOJ 4 1637-1644 [7 Mullet J E.Orozco. E M . J r a n d C h u a N-H (1985) Plant Mol Biol 4 39-54 18 Crossland L D Rodermel S R and Bogorad L (1984)Proc NmlAead Set USA 81 406~b4064 19 Hanley-Bowdom L (1986) PhD Dnssertatmn The Rocke[eller Umverslty 20 Hawley D K and McClure W R (198"~) ,~uclewAcids Res I I. 2237-~55 21 Kung S D and Lm C M (1985)Nuclew

Multiple mechanisms of neutralization of animal viruses l',ligel J. Dimmock Anawral annbod:es play a malor role m protecang anvnais f r o m mfect~n by viruses, but the mechamsm o f neutrahzaaon ~ not necessardy as sunple as u was once thought to be Nezarahzanon can be qt~ahtanvely affected by mleracaons between the virus, the cell receptor and the class o f tmmunoglobuha, and :t seems hkeh, that any early f u n c ~ n requ:red f o r the establtshnwnt o f refection ts vub~erable to the acnon o f anl~body N e u t r a h z m g ant,body (produced by B cells) and cell-medmted immune responses (winch depend o n T cells) provide immunity agamst remfectmon by viruses and c o n t n b u t e to recovery from p n m a r y refection H o w e v e r the two systems are so mtertwlned that it msdifficult to d e t e r m i n e the part each plays in comb a t t m g a particular refection, a l t h o u g h usually o n e o r the o t h e r plays the pred o m m a n t role Immunization estabhshes protective immumty w~thout the mdlvmdual haxang to suffer the natural disease a n d the efficacy of a vaccine for reasons of technical slmpllcRy has traditionally been assessed by m e a s u n n g the neutralnzmg antibody response Tins. o n the whole, correlates well with protection people or ammals with a Ingh response do not get remfected HowN J Dimmock Is at the Deparanent of BIologwal ~;cwnce~. Umverstl~, of Wantttk Coventry. CV4 7AL UK

ever, it is n o w b e c o m i n g pessl01e to assess, at least o n a hmRed scale, the Tcell response to nmmum7atmon a n d thus to evaluate the relative importance of B and T cells m protection agamst disease This review focuses o n neutralBatlon winch ns the inactivation of v~rus mfectwnty by a n t i b o d y alone, a n d m particular on the m e c h a n i s m by winch neutrab7atlon ms b r o u g h t a b o u t Nonetheless, mt should not b e forgotten that both neut r r h z m g and n o n - n e u t r a h z l n g antibodies (see below) can also mactwate infectivity m conjunction with accessory factors such as c o m p l e m e n t and antibodyd e p e n d e n t cytotoxmc cells This may seem an o d d subject for a c o n t e m p o r a r y review as. for m a n y years, mt was firmly held that neutrahzatmon resulted from antibody blocking the a t t a c h m e n t of virus to Its host cell - or in today's jargon the a t t a c h m e n t of t h e virus a t t a c h m e n t protem ( V A P ) to a cell receptor u m t ( C R U ) Now we k n o w that at best this ms

.~JIgIt7 El..c,.nerSoencePuhln~hcr~B V Amsterdam 0376- s(IbT.h"//${~IX}

Acids Res 13 7543-7549 22 Apel, K and Bogorad L (1976) Eur J Btochem 67. 615-620 23 Tewan K K andGoel A (1983)B:ochem~~ ' 22. 2142-2148 24 Lerbs S. Brautlgam E and Parthier, B (1985) EMBO J 4. 1661-1666 25 Grmssem. W. Greenberg B M . Zurawskl G and Hallnck R B (1986)Methods Enzymol 118.253--270 26 Orozco E M Jr. Mullet. J E . HanleyBowdoin L and Chua. N-H (1986)Methods En~'mol 118 232-253 27 Greenberg. B M. Gruissem W and Halhck R B (1984) PIm.t Mol Btol 3.97-109 28 Sttrdnvanl. S M Crossland, L D and Bogorad. L (1985) Proe NatlAcad Set USA 82.4886--489O 29 Krebbers. E T . Laumnua. I M. Mclntosh L and Bogorad. L (1982) Nudew Ac:ds Res I0, 4985-5002 "~0 Zurawskt G Petrol B , Bottc, rnley, W and WhRfeld. P R (1981) Nudew Acids Res 9 3251-3270 31 Zurawskn. G Bohnert, H J . Whltfeld P P and Bottomley W (1982) Prot ? ' ' A c a d Set USA 79 7699-7703

only part of a story which is currently b e m g unravelled n 2 In c o n s i d e n n g n e u t r a h z a t l o n in general nt is a p p a r e n t t h a t the m e c h a n i s m d e p e n d s on p r o p e ~ e s i n t n n s l c to the virus, the a n u b o d y a n d the host cell (Fig l ) (1) T h e o u t e r p r o t e m s of most animal vwuses bear o n t h e i r external surfaces specific neutralization sites a n d only w h e n antibody a t t a c h e s to these does neutrahzatlon result A corollary to tins is that a t t a c h m e n t o f a n t i b o d y to any o t h e r anttgemc site, w h e t h e r o n the same or a different surface protein, does not neutralize This ns n o n - n e u t r a h z m g anUb o d y in a different c a t e g o r y are a few viruses hke African swme fever winch c a n n o t be n e u t r a h z e d a l t h o u g h they are capable of ehcntmg anti-viral i m m u n o g l o b u h n s 2 They m a y lack neutralization sites but It ns conceivable t h a t they have such sites winch for s o m e reason are not immupogemc (2) C u r r e n t evidence suggests that neutrahzatnon can a b r o g a t e any o n e of a n u m b e r of early e v e n t s nn infectmn , w h i c h operationally starts with attachm e n t of vwus to a cell a n d e n d s vath w h a t e v e r event p r e c e d e s the first wrusdirected macromolecular synthetic e v e n t This mcludes b l o c h n g of the a t t a c h m e n t of a V A P to a C R U mentnoned a b o v e but tins is only o n e of m a n y mechanisms (3) O n e e m e r g m g vmw is that neutral~zat~on can result f r o m c o n f o r m a t l o n a l restrmnts Imposed u p o n wral proteins by neutrahzlng a n t i b o d y ( r e w e w e d tn R e f

T I B S 12 - F e b r u a r y 1 9 8 7

Some v,ruses have neutral;zat,onand non-neulrahzat;onproteins the former havelboth neutrahzat~onand non-neutrahzat~onant=gen=cs,les D~tferent sttes may med~ate~neutrahzat~onm d=fferentways

71

mg glycophonn 5) from making contact with the v~rus attachment s~te (VAS) on the tip of the haemagglutmln VAP l,hough the slahc acld-beanng CRU on VIRUS PROTEIN cultured cells has not been ~dentlfied, speculaUon suggests that tins ,s a ,.~olecule long enough to reach through the fringe of lgG molecules to the VAS Obviously physical interference with attachment becomes more stgmficant when virus Is neutrahzed by the larger dlmenc secretory (s)lgA and pentamenc IgM molecules6.7 A more subtle and complex interacCELL --" •" - C L A S S O F Ig tion between CRU, VAS and antibody ,s suggested by the data on the neutralD,Iferent cells have ddferent CRUs Different physical properties of Batmn of entermarus 71 winch show that which may/may not recognize a monomenc (IgG and IgA) and polymenc both the kinetics and extent of neutralvmon--lg complex and hence ankbodles (slgA and IgM) affecl the delermlne n~' ~trahzatlon mechanism o! neulrahzal~on izatlon depend upon the cell m which residual mfectwlty is assayeds (Fig 3) Fig ! Fac~ors,mportantmdewrmm,ngthemeclum~smofneutrahzauon Alteranonofanyonemayquahla. One explanahon for the increase m new nvely alter the mechamsm trahzat,on mzght be that new ant,gemc 2), they may induce novel conforma- The cell's inllueace tat neutralization sRes occur eRber at the Interface tional changes or prevent conformaWhen the bunyav~rus La Crosse reacts between the VAP ann CRU (Fig 4a) or ttonal rearrangements that are necessary with monoclonal antibody 3G8, tt ~s neu- by an allostenc mod=ficatlon of the VAP prereqmslte for successful mfecuon trahzed If assayed on hamster (BHK) following Rs interaction with the cell One example of the former is 'synergistic cells but retains full mfectwRy for mos- (Fig 4b) Eitber way anUbody can only neutrah~tton' resulting from increased quito cells, for a ~econd monoclonal anti- brad to wrus and neutrahze ~t after the bmdmg of a second anUbody following body, 2B9, the reverse is true 3 Clearly in v~rus has interacted w~th the cell It the earher attachment of another anti- th~s example it ts the cell that determines would be relanvely simple to test this whether or not the vmas ts neutralized ,dea by remowng free anUbody before body molecule to a different epttope (4) The class of antibody also deter- but unfortunately we know nothing cells are inoculated but this has not yet about the wrus--cell interactmn, not even been done mines the mechanism of neutrahzatlon From the foregoing it is clear that the (5) The cell, through the properties of whether sarus brads to both cells or is the CRU it bears on its surface ~s also a unable to brad to the cell for which it ~s CRU can play a key role m determining determining factor in the mechanism of neutrahzed An explanation is suggested neutrahzatlon ldennficatlon of CRUs is by the fadure of neutralized influenza difficult as there ~sno certainty that every neutralization (Fig. 1) (6) Imposed upon the foregoing are virus to bind to chicken erythrocytes type of cell surface molecule to which a quantitative constderauons relating to (R P Possee, PhDThesls, UmversRyof v,rus brads is a true CRU in the sense of the number of antibody molecules per Warwick, 1981) even though the same leading to a productive refection A betvirus p a r a d e required to elicit neutrah- neutralized virus brads with normal kine- ter approach is to use monoclonal anuzation. Kinet¢ data have long shown tics to clucken embryo fibroblast, BHK, bodies which bind m a specific cell surthat neutralization is a first-order reac- mouse L and human foreskin fibroblast face component and block mfectwtty tion, but ~t has not been explained how cells in culture 4 The receptor for The antibody can then be used as a preone lgG molecule (approximately the influenza wrus is linked to a vanety of parauve affimty reagent to punfy the same s~ze as one of the 500-1000 cell surface molecules vm a terminal CRU What =s known ,s that CRUs can haemagglutmm sp~kes in an influenza stahc acid residue Figure 2 suggests that be carbohydrate (term,nal slahc acid v~rus particle) could mactwate the mfec- the &fference in binding might result for influenza wrusesg), hpld (phosfrom the relatwely large neutralt~ng IgG phandylsenne for vesicular stomatms tlwty of such a large muluvalent hgand Recent unpublished data from my lab- molecule preventing the shorter erythro- vwus '°) or prote,n (l~-adrenerg,c receporatory w3th monoclonal anubod~es cyte CRU (probably the s,ahc acid-bear- tor for reowrus 't and an un,denttfied 90 (H P Taylor and N J Dnnmock) have spotlighted the old sRuat~on once again and remove the argument that the firstorder Ionetlcs ~ ere an artefact of the -" 16nm paratope (binding site of the ant,body) heterogeneity of polyclonal antlsera 14rim With so many vanables there is unlikely to be any common mechanism 5nm CRU " - ~ " - ? / / / ~ / / / / / , , T / of neutrahzat~on, except ,n the most general of terms A discussion of how each / / / / / / / / / / / / / / of the major factors- cell, wrus and antv BHK, CEF, HFF, L rbc body - affects neutrahzauon now follows Throughout this article, unless specified otherwise, 'antibody' refers to F~g 2 Hypolltencal scheme to explain hot* IgG block~ attachment of influenza ttnts to erwhroc~ te~ hut not other cells lgG

TIBS 12-February 1987

72

100

A

O

~t~ GMK

'~ o

o~IO O

0

N L_ m

RD c i

I

10 30

I

60

I

120

Tirne(mm) Fig 3 Effect of the cell o n neutrahzauon of en. teroHrus 71 After reatuon with annbody, the mixture was spht and assa)ed on monkey (GMK) or human (IOD) cells Adapted from Ref 8 with permISSIon

kDa polypeptlde for rhndovlruses 12) There ts gro~mg evidence that cell surface components of the ~mmune system which have receptor functmn (la antP gen 32, CR2 receptor for complement component C3d 33 and T4 ~) and those whuch act as receptors for the chemical transmnters adrenahn n, acetyl chohne 35

haemaggluttmn, H A ) but not another (the neuramlmdase) 14 Tins wins also demonstrates that only a Inmted number of sutes on the H A (four per H A monomer) are neutralBmg is and that attachment of antibody to other antv gemc sutes on the H A does not cause neutrahzaoon 16 Such non-neutralmng antubody is well known but the sites have not been physically mapped It is noteworthy that the VAS Itself, centermg on amino a o d residue 226 (Ref 17), and located at the tnp of each monomer, IS apparently not immunogemc, possubly because ut hes m a depression and us not easdy seen by the unmune system The same argument apphes to the VAS of p¢omawruses ~s The exclusion of anubody from the VAS itself is entirely conslstent vdth the ummpeded binding of neutrahzed influenza wrus to cultured cells (see above) The relatwe concentrations of v~rus particles and antibody us an additional factor m neutrahzat~on since, under condmons which allow them to form aggregates, even non-neutrahzmg antibody can decrease infectivity It ts difficult to deterrmne nf aggregation us important m vtvo as it depends on the proportuon of VLrUSand antnbody present For instance, at a hugh antnbody vwus ratno aggregatuon does not take place, as all avanlable sites are saturated and crosshnlung of partucles cannot occur However, one monoclonal IgG to polnovirus appears to

(a)

iI I

iU

m

•-- new antigenic rote

and epndermal growth factor36, also serve as CRUs for certain speclfuc vuruses One can also surmnse that factors affecting the presentatuon of CRUs such as the flunduty of the surrounding membrane or the detauled anatomncal structure of cell (e g wlln, culua)may affect the binding of vnrus to cells and hence neutrahzatLon Thus ut should be borne un mind that mechamsms of neutrahzauon of vwuses unfectmg dufferentlated cells of the appropnate target tissue m vtvo may be very different from those operatnng when cultured cells un the laboratory are used Indeed tlus may be the reason why some antnbodues that neutralnze m vztro are non-protectnve un annmals la Viral characteristics affecting neutralization Influenza v~rus well illustrates how neutrahzatuon can be meduated through one vmral surface protein (the

neutralize only by aggregation 19 and all other neutralmng ant~bodnes tested also formed aggregates with reduced mfectlvtty20 Polymenc antnboch~are no better than monomenc at causmg aggregation. Various early unteracaons of vu'us and cell can be unlubuted by antibody thus causing neutrahzatnon (Table I). lnhzbmon of attachment Monoclonal antibody to reovlrus t~l protein, ~hlch IS located at each vertex of the ncosahedral wnon, us neutral~.mg and data suggest that ut blocks attachment 21, prowdmg one example of the traditional v~ew of neutrahzatlon However, the quantitative relationslup between neutrahzatmn and the fadure of the vn'us to attach has not yet been estabhshed How attachment us blocked is not known but three posslblhhes are shown m Fig 5 lnhlbazon of penetrauon By analogy with receptor-mediated endocytOSlS, penetration of virus Is thought to occur by the nucleaUon of a cnOcal, but unspecified, number of CRUs to form a cell receptor site (CRS) (Fig. 6a). Here antnbody would pernnt attachment but would jam the 'zipper' mechamsm and prevent endocytosns and penetration Attachment without penetraUon occurs when influenza vlrns Is neutrahzed by the polymenc anhbodnes slgA and lgM 6,7 lnhlbmon of uncoaung Uncoatmg of many enveloped and non-enveloped vuruses unvolves co,fformauonal changes m the external proteins wheh are

CRU-~~ !!

,

U

I

(b) I

VAP"->'~LJ

new anhgenlcsite formed

CRU_~.~J~L"! I I

I I

Fig 4 Possible explanauom oj the cell-dependent neutrahzaaon of enterovlrus 71 (see Fig 3) through the creat¢on of new anngemc sttes etther (a) directly or (b) allostencally

TIBS 12- February 1987

73

Taule l Posstble mechamsms of neutrahmnon

(A) Antibody reacl,ng with a single virus particle neutmhzes mfectiwty by Inlnb~Ungattachment Allowing auachment but mh~b~tmgpenetration Allowing penetrauon but mhtbmng uncoat, ng Allowing uncoal,ng but lnhlbmng a later funcoon (B) Antibody reacting with two or more virus particles neutralizes by aggregation

tnggered by the low pH enwronment of the endocytotlc vesicle22and it would not be too surpnsmg if the binding of antibody to particular sites abrogated these Some, but by no means a!!23,24, plcornawrus anubodles induce conformaUonal changes recogmzable as different charged states and Mandel argued 25that anubody froze the virus m the conformation (Fig 7) which stdl allowed virus to attach to cells, penetrate and uncoat but resulted m degradation of the viral genome It appears that crosshnlang of ~dentlcal anugemc rotes on the adjacent faces of the icosahedrnl ,arus is essenttal for neutrabzauon as cleavage of the annbody to monomenc Fab umts by papmn restores both mfectmty and the pl from an acid value back to neutrahty 26 However, the Mandel model has recently been challenged due to a lack of proportlonahty between the pl shift and neutrahzauon by polyclonal rabbR antiserum in parucular 23, and needs to be re-evaluated In other c~rcumstances antibody n a g h t stabdize the capsld making tf refractory to those conformatmnal changes which are necessary for uncoating or alternatwely create changes which hkewise abrogate uncoatmg A recent example is neutralized West Nile viru~ (an enveloped flawvirus) which is taken up into vesicles but fails to undergo its normal uncoatlng by fusion 27

lnhlbmon of subsequent stages The lonetlcS and end result of the early stages of refection of influenza vu'us neutrahzed with IgG are mchstmgulshable from those of mfecttous virus2s Neutrahzed virus undergoes pnmary uncoating, losing its hlad envelope, and as genome accumulates m the nucleus apparently intact. However, there is no primary transcnptmn To explain these observaUons It was suggested that an antibodyreduced signal is conveyed across the viral envelope and results m the fmlure of transcnpuon However, more recent evidence shows that the genome of neutrahzed virus m the cell has much greater RNase resistance than infectious wrus favouring the idea that, rather than a chrect effect on transcnptmn, there is a fadure m secondary uncoatmg of the viral core and the genome ~s not released in a transcribable state (R Ragg and N J Dlmmock, unpubhshed)

The role of antibedy in neutralizalian

Class of lmmunoglobuhn The monomenc Immunoglobuhns IgG and IgA, the dlmenc IgA and secretory sIgA and pentamenc IgM are all neutrahzmg Thetr various physical structures suggest that they might bring about neutralization by different mechamsms In support of this the best data compared neutrahzatlon of antiinfluenza slgA wRh IgA denved from it by differential reductton 6 Neutrahzlng monomeric lgA behaved exactly hke IgG (above) and fmled to affect any of the early stages of infection In BHK cells However, the v3rus neutrahzed by slgA, about 50% fmled to attach to cells and 50% attached but faded to enter At this stage we do not have the mformaUon to decide ff physical properties or valency are responsthle for the different modes of acOon of lgA and slgA. lgM neutrahzed influenza exactly like slgA, perhaps indicating a common neutrahzatlon meehamsm 7

Valency Neutrahzauon of poho~nrus by antibody of certain speaficmes reqmres crosshnkang by bivalent antibodye6 but F a b I fragments of neutrahzmg lgG polyclona129 antibody to influenza HAa have neutrahzmg acuvrty Thus, depending on the system, neutralization may or may not involve crosshnlang of epltopes of the vwus parUcle 2

Quantuanve aspects Maxtmum number of antibody molecules/pamcle Data from our laboratory using 12SI-labelled antibody of known specafic acuvity show that influenza wrus m solution saturates ruth

DIRECT

INDIRECT

only one of the three potential binding sites on each HA spike occupied by antiHA lgG The same ~s true of polyclonal lgG where, assuming all neutrahzatlon sites to be equally immunogemc, one antibody molecule is bound per 12 potential binding sites, and vath polyclonal neutralmng slgA one molecule binds per two HA spikes Thus It seems that stenc constraints hmlt the packing of antibody on native influenza wrus to one four-chmn ~mmunoglobuhn unit per HA spike However, virus attached to plastic binds three monoclonal IgG molecules per spike suggesung that it has undergone conformatlonal changes which affect ItS interaction vath antibody30

Mtmmum number of annbody molecules neededfor neutrahzanon This contentious Issue stems from kme[ic neutrahzatlon data which show that about one or a few IgG molecules can inactivate one infectious virus particle Clearly it ts Impossible to account for neutrahzatlon of a wrus such as influenza wtth 3000 VASs by a mechamsm such as inhlbRion of attachment, but the general argument advanced through this article that antibody can trigger a conformatlonal change which is lethal to the virus would explmn the data Now that monoclonal antibodies are available to eliminate the heterogeneity inherent m neutralmng polyclonal IgG, has the situation changed~ Firstly, our kinetic neutrahzatlon data (H P Taylor and N J Dimmock, unpublished) show clearly that one or two hits (molecules of igG) onlyare required forneutrahzatlon Secondly, we calculate that when 50% of a populauon of influenza virus ,s neutrahzed there are 50 IgG molecules per particle Thus there is a major discrepancy How can it be resolved~ We suggest that while the monoclonal antibodies and HA spikes each represent a homogeneous population of ideuttcal molecules with respect to structure, HA spikes are not homogeneous with respect to the underlying v~ral core We suggest

ALLOSTERIC

VAP

CRU

Ftg 5 Possible mechamsms by whwh altachment of wrus to the CRU Is mhlbrted by annbodv

T I B S 12 - F e b r u a r y 1 9 8 7

74 (a) ENDOCYTOSIS PLASMA MEMBRANE(INSIDE)

CRU

NUCLEATION ~,

.

, , ~ / - CRS

J,

J,

that there are 20 H A molecules which commumcate m some umque way with the core structure m such a way that neutrahzatlon can be effected by an antibody molecule Inn&rig to them Bmchng of an antibody to any of the other 980 H A spikes wdl not neutralize. Thus a single antibody molecule c a n neutralize a virus pa,'ticle by binding to one of the 20 'neutrahzation relevant' H A sp,kes but many more antibo&es wdl brad to the more numerous 'neutrahzation irrelevant' H A molecules By such a hypothesis the apparent discrepancy between kanetic and biochemical data could be resolved Finally. the fact that relaUvely small amounts of neutrahzlng antibody can attach to wrus without causing neutrahzatlon reminds us that the antibody Itself can act as a V A P and potentiate refection under some c~rcumbtances3~ W h z e h p a r a t o p e s are p r e s e n t m vivo ~

(b) NEUTF:IALIZATION

Studms using monoclonal antibodms, particularly to pohovlrus, have shown that IgGs wzth dLfferent paratopes effect neutrahzation by &fferent mechanisms23 24 In th~s regard the mechamsm of

1

NUCLEATION PREVENTED

neuzrahzation m man, the natural host, wdl depend upon those paratopes that are most abundant and/or have the hzghest affinity Such analyses of the dzstnbutlon of paratopes m v t v o are necessary before we can understand how neutrahzation takes place m v w o

Conclusion Regrettably there seems to be no general mechamsm of neutrahzation for any one virus neutralizaUon Js determined by the znteractzon of virus, antibody and ceg. What emerges is the need for more mformauon, particularly about Fig 6 Scheme suggeslmg how amlbod~ tould neutrahze wrus tb) by preventing the normal nucleauon of neutrahzatlon m the natural host Pascell receptor units ( CR Us) to form a funcuonal cell receptor sue ( CRS ) fa ) swe protection data show that only some neutralmng anubodles, even though ag have the same lgG subclass, protect antreals from mfection. It seems hkely that the key to the puzzle hes gqth the target cell and the realzzation that data relevant to neutrahzaUon m v t v o can only be obtained by stud3ang refection of the dff• ferentzated target tissue

noQnbody __k

Acknowledgement I am very grateful to Howard Taylor and ~ c h a r d Pdgg for pernusszon to czte their unpubhshed data

+ mobs of certo|n spec,flctty in f e c t i o u s

non- infect,ous

Frg 7 Neutrahzatlon of Type I pohovlrus by preve,mon of penetration as a result of'freezing" the virus m one o f two charged conforma~ons Antthod~es of other speccficmes neutrallze m orher way~

References I Mandel.B (1979)Compr Vtrol 15.37-121 2 Dtmmock, N J (1984)J Gen VIrol 65, 1015-1022 3 Gra'4y, L J and IOnch, W (1985)J Gen V~rM 66, 2773--2776 4 Dtmnmck. N J. Taylm. H P and Carver.

TIBS 12- February 1987

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A S (1984) In Segmented Negauve ,Strand Vu'uses (Compans, R W and Bisbop, D H L , eds), laP 355-359, Acadenne Press Vutala, J and Jarnefelt, J (1985) Trends Btochem Set 10,392-395 Taylor, H P and DImmock. N J (1985) J Exp Med 161,198--209 Taylor, H P and I~mmock, N J (1985) ./ Gen Vtrol 66.903-907 KlelI~n,L (1985)3 Gen Viroi 66.2279-2283 Gottschalk, A (1958) Adv En~mol 20, 135-146 Schlegl, R , Tralka. T , Wflhngham, M and Pastan, ! (1983) Ce//32.639--646 Co. M . Ganlton, G N,Fields. B N and Greene, M J (1985)Proe Nail Acad S~ USA 82. 1494-1498 Tomasstm. J E and Colonno. R J (1986) J VIrol 58. 290-295 Buchmeler. M J . Lew~clu, H A , Talbot. P J and Knobler, R L (1984)Virology 132. 261-276 Webster, R G andLaver.W G (1967)./ immunol 42.49--55 Wdson. I A SkeheI. J J andWdey, D C

(1981) Nature289, 366-373 16 Breschlun. A M . Ahem. J and White. D O (1981) Virology 113. 130-140 17 Rogers, G N , Paulson, J C , Darnels. R S . Skehel J J . Wdson. I A and Wiley, D C (1983) Nature 304.76-78 18 Ro~man. M G , Arnold. E . Enekson. J W . Frankenberger. E A , Gnf-fith. J P . Hecht. H , Johnson, J E . Kamer. G . Luo. M . Mosser. A G , Rueckert, R R . Sherry. B ar, ! Vnend. G (1985) Nature 317, 145-153 19 Thomas. A A M . Bnoen, P and Boey~, A (1985)J Vtrol 54,7-13 20 Thomas. A A M , Vrllsen, R and Boeye. A (1986)3 Vtrol 59,479--485 21 Lee, P W K , Hayes. E C and Jokhk W K (1981) Virology 108, 156--163 22 Patterson, S and Oxlord, J S (1986) Vaccine 4.79--90 23 Bnoen, P , Tl'.omas. A A M and Boeye, A (1985)./ Gen Vtrol 66.609-613 24 lcenogle, J , Sluwen, H , Duke, G , Gilbert, S , Rueckett, R and Anderegg, J (1983) ./ Vu,oi 127, 412-425 25 Mandel B (1976)Virology69,500-510

26 Enum, E A , Ostapchuk. p and Wtmmer. E (1983)J Vtrol 48,547-550 27 Golhns, S and Porter[ield J (1986) Nature 321. 244--246 28 Possee. R D . ScluId. G C and Danmock. N J (1982)J Gen Vtrol 58.373-386 29 Lafferty, K J (1963) VtroloD' 21. 76-90 30 Nesterovdcz A . Laver, W G and Jackson. D C (1985)./ Gen Vtrol 66. 1687-1695 31 Pems. J S M and Porterfield, J S (1979) Nature 282.509-511 32 Inada. T and Mnns, C A (1984) Nature 309. 59-61 33 Fmgeroth, J D . Wels, J J . Tedder. T F . Strormnger J L , Biro A P and Feardon D T (1984)Proc Nail Acad Set USA 81. 4510--4514 34 Klalzman, D and Gluckman, J C ( 1 9 8 6 ) I m muno/ Todoy 7. 291-296 35 Lantz. T L,Burrage, T G . S n n t h , A L , Cnek, J andTignor, G H (1982)Scwnce215 182-184 36 Eppstem. D A , Marsh, Y V , Schrelber, A B . Newman, S R . Todaro, G J and Nestor.J J 0985) Nature 318,663--665

Polyphosphoinositide phosphodiesterase: regulation by a novel guanine nucleotide binding protein, Gp

glycerol Is the activator of protein kmase C (Ref 1) Thus, cellular responses (e g contracnon, secretion or cell growth) can possibly be explamed on the basis of receptor-mediated activation of PPl-pde and the resultant second messengers generated Ca 2+ and dlacylgly_cerol, hke cAMP, activate specific protein kanases responsible for protein phosphorylatlon ~vinch then control the Intracellular machinery of the cell

5 6 7 8 9 l0 It

12 13

14 15

Shamshad Cockcroft A novd guanine mJd~ade bmdmgprot~n, G~ may be revolved m couplingreceptoracavatton to the breakdown of phosp~hdylmosuol 4,5-ba'plunph~ by a phogdguttesterase Ttus generates two second messengers, mosttol 1,4,5,-tnphosphate and dtacylglyceroi The stimulation of receptors by hormones, neurotransrmtters and growth factors is fundamental to the response of cells to external sttmnlation. V/inlst there are a great variety of external agnals only a hmlted repertoire of second messengers are employed w]thm cells The first second messenger to be identified was cychc AMP and the mechamsm of the cAMP generating system is now fairly well understood, the agnmst-receptor complex interacts with a guanine nucleoade bm&ng protein (G o and tins m turn actwates adenylate cyclase Another G protein, G,, has been identified and tins mediates mlublUon of adenylate cyclase A second signal transductlon system that has received a lot of attenUon recentl,, uuhzes an mosltol-contammg pb,,sphohpid, S Cockcro]t rs at Ih, Department of Expenmenud Pathology. School of Medicine. Unwersuy College London. UmversttvSt. London WCIE 61J. UK

phosphatldyllnosltol 4,5-insphosphate (PlP2), it generates two intracellular agrials simultaneously Tins transmembrane signPIhng system regulates the concentration of Ca 2+ m the cytosol and controls the acUvRy of protein kmase C Its components, winch have been defined only recently, appear to bear stnkmg resemblance to the famJhar adenylate cyclase system. Tins article is mainly concerned with how the catalytic umt of the mositol hpld qgnalhng system interacts with receptors wa a gnamne nucleotlde binding protein termed Gp Polyphosphomo'~ trade phospho~iesterase (PPl-pde, a phosphohpase C) is the catalytic umt that hydrolyses the mosltol-contammg phosphohplds to generate the water-soluble mosltol phosphates and dlaq,glycerol Of the several mosltol phosphates formed, only mosxtol 1,4,5-tmphosphate acts as the mtracellular messenger mobdmng Ca 2+ from the endoplasnuc renculum whilst d]acyl-

ldenlifw.afion of the enzyme involved in signal Iransduetion Pnor to 1981 it was thought that PI (phosphatldyhnositol) was the ]mttal substrate hydrolysed dunng receptor stimulation PIP (phosphatldyhnosttol 4phosphate) and PIP 2 are sequentially formed by phosphorylatton of PI by two separate kmases which are located m the plasma membrane [t is now clear that of the three Jnosltol hplds4 PIP 2 (and possibly PIP) ]s the mltml target for the PPI-

pde A cytosollc Pl-pde was discovered by Dawson in 1959 and since then it has been found m every mannnahan tissue ,n winch It has been sought, as well as in ingher plants and some bacteria -~ Although all the early mvestlgatlons studied the acav~ty of the soluble enzyme using Pl as a substrate, it has been demonstrated recently that this enzyme Is able to hydrolyse all three inosttol phosphohplds with equal faclhty3 Its activity has normally been assessed using pure lipid substrates, ,a not unai monolayers made of PI or rmxtures of hplds were studied was it found that the packing of the hplds is crucial in deter-

1987 ElsevzerSoen~Pubhsher~B ~ Amslerdam0.t76-'~0~7r87/$0200